PROJECT SUMMARY Pluripotency is controlled by key transcription factors such as Nanog, Oct4, and Sox2 in conjunction with many epigenetic regulators including DNA (de)methylation enzymes, which together form the interconnected pluripotency regulatory networks. Mouse pluripotent stem cells exist in two distinct stable pluripotent states (naive and primed), which are represented by ground state embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), respectively. Human ESCs share defining features of primed pluripotency with mouse EpiSCs, and naive human ESCs/iPSCs can also be established, although our understanding of naive and primed pluripotency of hESCs is very limited. In pursuit of a deeper understanding of pluripotency under defined nave and primed culture conditions, we have recently discovered Zfp281 and Tet1/2 as critical players for controlling pluripotent states in vitro. While in vitro studies are instrumental, it remains critical to confirm whether the insights they provide are relevant to events taking place in vivo during epiblast maturation from naive pre- implantation blastocyst to the primed post-implantation epiblast. We will employ our integrated genomics and proteomics approach combined with in vivo mouse models to dig deeper into mechanism in this application with a broader effort to dissect novel regulatory mechanism by which Zfp281/ZNF281 orchestrates transcriptional and epigenetic processes in controlling primed versus naive pluripotency in both in vitro cellular model and in vivo mouse knockout models. We will test the hypothesis that the pluripotency factor Zfp281/ZNF281 plays critical roles in coordinating opposing functions of Tet1 and Tet2 and the crosstalk between Tet1/2 and DNMT3a/3b proteins for transcriptional and epigenetic control of pluripotent states in both mouse and human systems. Our proposed studies encompass the following three Specific Aims. 1) Define the functional connection between Zfp281-DNMTs in controlling pluripotent states. 2) Define the functional connection between Zfp281 and TETs for primed pluripotency. 3) Investigate transcriptional and epigenetic regulatory roles of Zfp281 in epiblast maturation of mouse early embryos. Our work represents a conceptual advance and a fundamental contribution to the understanding of early mammalian development, which is grounded by pluripotent state transitions. Our combined approach to study Zfp281 with both cellular and mouse models of pluripotent state transitions will provide fundamental knowledge on finely coordinated transcriptional and epigenetic control of mammalian cell fate changes and embryonic development, dysregulation of which is often associated with disease and cancer.